The present invention relates generally to implantable biomaterials prepared by a rapid technique from aldehyde-treated autologous connective tissues, and particularly to such medical devices as tissue heart valves, patches, conduits and other intravascular or intracardiac prosthetic devices which possess improved biocompatibility and anticalcification properties.
Biomaterials prepared from glutaraldehyde crosslinked heterologous connective tissues such as bovine pericardium or porcine aortic valves are widely used in reconstructive and replacement procedures such as cardiovascular surgery in the form of bioprosthetic heart valves, valved or non-valved conduits, fashionable patches and the like. Stabilization of tissues with glutaraldehyde improves their biomechanical properties, increases their resistance to collagenase and reduces tissue-induced thrombogenicity. See Nimni et al., J. Biol. Chem., 243:1457-1466 (1968). The alternative of using native, non-crosslinked tissues is disfavored because performance is limited due to tissue thickening and shrinkage, enzymatic digestion and subsequent loss of mechanical properties. See Ross et al., Ann. Thorac. Surg., 13:97-103 (1972). These reactions are characteristic of a tissue which is exposed to a new, non-physiological environment (such as pericardium exposed to flowing blood and extreme mechanical stress). Treatment of biological tissues with glutaraldehyde prevents these phenomenon by stopping cell metabolism and thus rendering the biomaterial usable in the new environment.
Glutaraldehyde, C.sub.5 H.sub.8 0.sub.2, contains two aldehyde groups and is commonly used in the fixation of biological tissues. Glutaraldehyde is a water soluble, reactive dialdehyde which when brought in contact with biological tissues penetrates fibrous connective tissues at a slow rate and interacts with terminal amino groups exposed by the tissue components. Several studies have shown that optimal crosslinking occurs after 5 or more days in the glutaraldehyde solution, while a short exposure (10-15 minutes) penetrates tissues only on their surface leaving a central core of suboptimally fixed structures. See Nimni et al., J. Biomed. Mater. Res., 21:741-771 (1987). The chemistry of glutaraldehyde crosslinking involves three main aspects of interest to this invention: (1) by means of the two reactive aldehyde groups, glutaraldehyde is capable of creating intra and intermolecular crosslinks, provided that the exposed amino groups are optimally spaced; (2) the second product of this reaction is glutaraldehyde which has reacted with only one amino group, thus exposing a free aldehyde moiety (unipoint fixation); and (3) glutaraldehyde polymerizes in time at neutral pH giving rise to polymers which are unstable and with time can release significant amounts of cytotoxic glutaraldehyde molecules into solution.
It is generally accepted that biomaterials prepared from tissues treated with glutaraldehyde for at least 14 days provide surgeons with biocompatible implantable medical devices with good long term durability. Still, several limitations of current devices indicate the need for improved biomaterials. See Schoen et al., Cardiovasc. Pathol., 1:29-52 (1992); Golomb G. et al., Am. J Pathol., 127:122-130 (1987).
The major limitation of current devices is calcification, the process in which organic tissue becomes hardened by the deposition of lime salts in the tissues. Calcification is the main limiting factor that affects the long term durability of glutaraldehyde treated tissues due to an interplay of a multitude of host factors (age, renal failure, hyperparathyroidism, mechanical stress) and implant related elements (glutaraldehyde per se, calcium binding matrix components, cellularity). The degree of calcium deposition is proportional to the degree of glutaraldehyde incorporation. It is thus believed that supoptimal fixation attained by a short term exposure to glutaraldehyde can induce less calcification, while maintaining the required mechanical characteristics.
Another limitation of current devices is variability. Variability in the clinical outcome has been attributed to the inherent variability in animal characteristics and tissue collection criteria such as age, race, nutritional state, sex, tissue quality, handling, cleaning, storage and transportation. Proper tissue selection requires consideration of a multitude of aspects such as collagen content, vascularity, cellularity, thickness and mechanical properties.
A third limitation of current devices is immunogenicity, the capacity to stimulate the formation of antibodies in a particular biological system. Although glutaraldehyde reduces considerably tissue antigenicity, a low-level of humoral (antibodies to collagen) and cellular (moderate leukocyte infiltration) immune response has been identified in animal and human subjects bearing these devices. Moreover, several studies have established a correlation between immune reactions and calcification.
Another limitation of current devices is viability, the ability to live, grow, and develop in the new biological system. Long term glutaraldehyde fixation protocols render tissues non-viable and prone to calcification--cells within the tissue are exposed to hypoxia (due to the delay between harvest and fixation) and after exposure the glutaraldehyde, devitalized cells are prone to calcification. Longer delays before completing fixation correlates with more pronounced calcification.
A fifth limitation of current devices is endothelialization in which free aldehyde groups derived from unipoint fixation and exposed towards the tissue surface inhibit endothelial cell coverage by the host.
Another limitation of current devices is cytotoxicity. Glutaraldehyde molecules may leach out, induce a local pro-inflammatory reaction and reduce tissue integration into the host organ. Simple repetitive washing in saline is ineffective in reducing cytotoxicity and pro-inflammatory reactions.
Another limitation of current devices is sterilization, the process of completely removing or destroying all microorganisms on a substance. The long procedure required for manufacturing of bioprosthetic tissues demands both careful bacteriological monitoring of each step and implementation of a final sterilization/storage step which may affect mechanical properties of the final product.
The present invention overcomes some of the above-mentioned limitations, without compromising the advantages gained by glutaraldehyde fixation. The major advantages of the proposed rapid glutaraldehyde fixation include: (1) the use of carefully selected autologous tissue will minimize variability and reduce the immunologic response; (2) the immediate fixation of autologous tissues will reduce the unwanted delay between tissue harvest and fixation, will warrant sterility and reduce the tendency to calcification, will limit glutaraldehyde polymerization and increase the possibility of saving a certain proportion of live cells, especially in the supoptimal fixed central areas; and (3) the development of a rapid glutaraldehyde neutralization procedure will ensure full depletion of deleterious aldehyde groups and leachable glutaraldehyde molecules thus providing with a non-cytotoxic and possibly endothelium-friendly biomaterial.